Hartmut K. Lichtenthaler

Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via a mevalonate-independent pathway

Isopentenyl diphosphate (IPP) is the biological C5 precursor of isoprenoids. By labeling experiments using [1‐13C]glucose, higher plants were shown to possess two distinct biosynthetic routes for IPP biosynthesis: while the cytoplasmic sterols were formed via the acetate/mevalonate pathway, the chloroplast‐bound isoprenoids (β‐carotene, lutein, prenyl chains of chlorophylls and plastoquinone‐9) were synthesized via a novel IPP biosynthesis pathway (glyceraldehyde phosphate/pyruvate pathway) which was first found in eubacteria and a green alga. The dichotomy in isoprenoid biosynthesis in higher plants allows a reasonable interpretation of previous odd and inconclusive results concerning the biosynthesis of chloroplast isoprenoids, which so far had mainly been interpreted in the frame of models using compartmentation of the mevalonate pathway.

Vegetation Stress: an Introduction to the Stress Concept in Plants

Summary This is a presentation of the essentials of the present stress concept in plants, which has been well developed in the past 60 years. Any unfavorable condition or substance that affects or blocks a plants metabolism, growth or development, is to be regarded as stress. Plant and vegetation stress can be induced by various natural and anthropogenic stress factors. One has to differentiate between short-term and long-term stress effects as well as between low stress events, which can be partially compensated for by acclimation, adaptation and repair mechanisms, and strong stress or chronic stress events causing considerable damage that may eventually lead to cell and plant death. The different stress syndrome responses of plants are summarized in a scheme. The major abiotic, biotic and anthropogenic stressors are listed. Some stress tolerance mechanisms are mentioned. Stress conditions and stress-induced damage in plants can be detected using the classical ecophysiological methods. In recent years various non-invasive methods sensing different parameters of the chlorophyll fluorescence have been developed to biomonitor stress constraints in plants and damage to their photosynthetic apparatus. These fluorescence methods can be applied repeatedly to the same leaf and plant, e.g. before and after stress events or during recovery. A new dimension in early stress detection in plants has been achieved by the novel high resolution fluorescence imaging analysis of plants, which not only senses the chlorophyll fluorescence, but also the bluegreen fluorescence emanating from epidermis cell walls which can change under stress induced strain. This powerful new technique opens new possibilities for stress detection in plants.

Photosynthetic activity, chloroplast ultrastructure, and leaf characteristics of high-light and low-light plants and of sun and shade leaves

The photosynthetic CO2-fixation rates, chlorophyll content, chloroplast ultrastructure and other leaf characteristics (e.g. variable fluorescence, stomata density, soluble carbohydrate content) were studied in a comparative way in sun and shade leaves of beech (Fagus sylvatica) and in high-light and low-light seedlings.1.Sun leaves of the beech possess a smaller leaf area, higher dry weight, lower water content, higher stomata density, higher chlorophyll a/b ratios and are thicker than the shade leaves. Sun leaves on the average contain more chlorophyll in a leaf area unit; the shade leaf exhibits more chlorophyll on a dry weight basis. Sun leaves show higher rates for dark respiration and a higher light saturation of photosynthetic CO2-fixation. Above 2000 lux they are more efficient in photosynthetic quantum conversion than the shade leaves.2.The development of HL-radish plants proceeds much faster than that of LL-plants. The cotyledons of HL-plants show a higher dry weight, lower water content, a higher ratio of chlorophyll a/b and a higher gross photosynthesis rate than the cotyledons of the LL-plants, which possess a higher chlorophyll content per dry weight basis. The large area of the HL-cotyledon on the one hand, as well as the higher stomata density and the higher respiration rate in the LL-cotyledon on the other hand, are not in agreement with the characteristics of sun and shade leaves respectively.3.The development, growth and wilting of wheat leaves and the appearance of the following leaves (leaf succession) is much faster at high quanta fluence rates than in weak light. The chlorophyll content is higher in the HL-leaf per unit leaf area and in the LL-leaf per g dry weight. There are no differences in the stomata density and leaf area between the HL- and LL-leaf. There are fewer differences between HL- and LL-leaves than in beech or radish leaves.4.The chloroplast ultrastructure of shade-type chloroplasts (shade leaves, LL-leaves) is not only characterized by a much higher number of thylakoids per granum and a higher stacking degree of thylakoids, but also by broader grana than in sun-type chloroplasts (sun leaves, HL-leaves). The chloroplasts of sun leaves and of HL-leaves exhibit large starch grains.5.Shade leaves and LL-leaves exhibit a higher maximum chlorophyll fluorescence and it takes more time for the fluorescence to decline to the steady state than in sun and HL-leaves. The variable fluorescence VF (ratio of fluorescence decrease to steady state fluorescence) is always higher in the sun and HL-leaf of the same physiological stage (maximum chlorophyll content of the leaf) than in the shade and LL-leaf. The fluorescence emission spectra of sun and HL-leaves show a higher proportion of chlorophyli fluorescence in the second emission maximum F2 than shade and LL-leaves.6.The level of soluble carbohydrates (reducing sugars) is significantly higher in sun and HL-leaves than in shade and LL-leaves and even reflects changes in the amounts of the daily incident light.7.Some but not all characteristics of mature sun and shade leaves are found in HL- and LL-leaves of seedlings. Leaf thickness, dry weight, chlorophyll content, soluble carbohydrate level, photosynthetic CO2-fixation, height and width of grana stacks and starch content, are good parameters to describe the differences between LL- and HL-leaves; with some reservations concerning age and physiological stage of leaf, a/b ratios, chlorophyll content per leaf area unit and the variable fluorescence are also suitable.

Detection of Red Edge Position and Chlorophyll Content by Reflectance Measurements Near 700 nm

Pigment contents was determined in and high spectral resolution reflectance measurements were acquired for spring, summer and autumn maple and horse chestnut leaves covering a wide range of chlorophyll content. Consistent and diagnostic differences in the red edge range (680-750 nm) of the reflectance spectrum were obtained for the various leaf samples of both species studied. This included the differences in the wavelength position of the red edge and in the reflectance values in the range of 690 to 710 nm. Both characteristics were found to be dependent on leaf chlorophyll concentration. The first derivative of reflectance spectra showed four peaks at 685-706, 710, 725 and 740 nm that were dependent in different degree on leaf age and pigment concentration in the leaves. The position and the magnitude of the first peak showed a high correlation with the leaf chlorophyll concentration. Reflectance at 700 nm was linearly dependent on the wavelength of the first peak. Variation of inflection point position with change in chlorophyll content was found small for yellow-green to dark green leaves (total chlorophyll in the range above 10 nmol/cm 2 ). Reflectance near 700 nm was found to be a very sensitive indicator of the red edge position as well as of chlorophyll concentration. The ratio of reflectances at 750 nm to that near 700 nm (R 750 /R 700 ) was directly proportional (correlation r 2 >0.95) to chlorophyll concentration. The ratio R 750 /R 700 as a newly established index for non-invasive in-vivo chlorophyll determination was tested by independent data sets in the range of Chl contents from 0.6 to more than 60 nmol/cm 2 of maple and chestnut leaves with an estimation error of Chl of less than 3.7 nmol/cm 2 .

How to correctly determine the different chlorophyll fluorescence parameters and the chlorophyll fluorescence decrease ratio RFd of leaves with the PAM fluorometer

This contribution is a practical guide to the measurement of the different chlorophyll (Chl) fluorescence parameters and gives examples of their development under high-irradiance stress. From the Chl fluorescence induction kinetics upon irradiation of dark-adapted leaves, measured with the PAM fluorometer, various Chl fluorescence parameters, ratios, and quenching coefficients can be determined, which provide information on the functionality of the photosystem 2 (PS2) and the photosynthetic apparatus. These are the parameters Fv, Fm, F0, Fm′, Fv′, NF, and ΔF, the Chl fluorescence ratios Fv/Fm, Fv/F0, ΔF/Fm′, as well as the photochemical (qP) and non-photochemical quenching coefficients (qN, qCN, and NPQ). qN consists of three components (qN = qE + qT + qI), the contribution of which can be determined via Chl fluorescence relaxation kinetics measured in the dark period after the induction kinetics. The above Chl fluorescence parameters and ratios, many of which are measured in the dark-adapted state of leaves, primarily provide information on the functionality of PS2. In fully developed green and dark-green leaves these Chl fluorescence parameters, measured at the upper adaxial leaf side, only reflect the Chl fluorescence of a small portion of the leaf chloroplasts of the green palisade parenchyma cells at the upper outer leaf half. Thus, PAM fluorometer measurements have to be performed at both leaf sides to obtain information on all chloroplasts of the whole leaf. Combined high irradiance (HI) and heat stress, applied at the upper leaf side, strongly reduced the quantum yield of the photochemical energy conversion at the upper leaf half to nearly zero, whereas the Chl fluorescence signals measured at the lower leaf side were not or only little affected. During this HL-stress treatment, qN, qCN, and NPQ increased in both leaf sides, but to a much higher extent at the lower compared to the upper leaf side. qN was the best indicator for non-photochemical quenching even during a stronger HL-stress, whereas qCN and NPQ decreased with progressive stress even though non-photochemical quenching still continued. It is strongly recommended to determine, in addition to the classical fluorescence parameters, via the PAM fluorometer also the Chl fluorescence decrease ratio RFd (Fd/Fs), which, when measured at saturation irradiance is directly correlated to the net CO2 assimilation rate (PN) of leaves. This RFd-ratio can be determined from the Chl fluorescence induction kinetics measured with the PAM fluorometer using continuous saturating light (cSL) during 4–5 min. As the RFd-values are fast measurable indicators correlating with the photosynthetic activity of whole leaves, they should always be determined via the PAM fluorometer parallel to the other Chl fluorescence coefficients and ratios.

Application of chlorophyll fluorescence in ecophysiology

SummaryIn vivo chlorophyll fluorescence measurements have become a valuable tool in ecophysiology. Fluorescence emission spectra are influenced by the reabsorption of the tissue and indicate the composition of the antenna system and are influenced by the chlorophyll content per leaf area. The fluorescence induction kinetics (“Kautsky effect”) can be used to study photosynthetic activity. These rapid, non-destructive methods can be applied for ecophysiological field research to check the vitality of plants and to document stress effects on the photosynthetic apparatus. The Rfd-values (Rfd=fd/fs), the ratio of the fluorescence decrease (fd) to the steady state fluorescence (fs), can be taken as a rapid vitality index of the leaves and trees. We here describe fundamental chlorophyll fluorescence results of leaves which are needed for the interpretation of in vivo fluorescence signatures in stress physiology and in the forest dieback research.

Cell wall bound ferulic acid, the major substance of the blue-green fluorescence emission of plants.

Summary When excited by UV-A radiation (e.g. N 2 -laser 337 nm), leaves of green plants exhibit, in addition to the red and far-red chlorophyll fluorescence, a genuine blue-green fluorescence emission with a maximum near 440–460 nm and a lower shoulder near 520–530 nm. Members of the Poaceae (monocotyledonous plants), e.g. maize ( Zea mays L.), wheat ( Triticum aestivum L.) or oat ( Auma sativa L.), possessed a much higher blue-green fluorescence emission than leaves of dicotyledonous plants, such as spinach ( Spinacia oleracea L.), tobacco ( Nicotiana tabacum L.), sunflower ( Helianthus annuus L.), foxtail ( Amaranthus caudatus L.) or purslane ( Portulaca oleracea L.). In the measured dicotyledonous plants, the blue-green fluorescence was in a similar range or considerably lower than the red chlorophyll fluorescence. In contrast, in Poaceae and other monocotyledonous plants, the blue-green fluorescence was generally several times higher than the red chlorophyll fluorescence. By alkaline hydrolysis of cell walls it was shown that the members of Poaceae exhibit a several times higher content of the blue-green fluorescent ferulic acid, both on leaf area and on a dry weight basis, than dicotyledonous plants, such as spinach, foxtail and purslane. Ferulic acid, being covalendy bound to the cell wall, appears to be the main emitter of the blue-green fluorescence of leaves which has been documented by several complementary observations. Other cell wall bound cinnamic acids (caffeic acid, p-coumaric acid), which are present in some plants in low amounts, contribute very little to the overall blue-green fluorescence emission of leaves. This also applies to the extractable flavonoids and cinnamic acids of the vacuole. The high fluorescence yield of ferulic acid containing cell walls is also documented by fluorescence excitation spectra of isolated, dried cell walls before and after alkaline hydrolysis of ferulic acid. Plants which do not possess ferulic acid in their cell walls, such as sunflower, pumpkin ( Cucurbita fici-folia L.) or tobacco, exhibit only a very faint blue-green fluorescence emission. Outdoor plants contained much higher levels of extractable flavonoids and cinnamic acids than greenhouse plants, yet the blue-green fluorescence was hardly modified indicating that the soluble flavonoids and cinnamic acids of the vacuole do not or only very little contribute to the blue-green fluorescence emission of plants.

The chlorophyll fluorescence ratio F735/F700 as an accurate measure of the chlorophyll content in plants

Abstract A remote sensing technique is presented to estimate the chlorophyll content in higher plants. The ratio between chlorophyll fluorescence at 735 nm and in the range 700–710 nm, F735/F700 was found to be linearly proportional to the chlorophyll content (with determination coefficient, r2, more than 0.95), and, thus, this ratio can be used as a precise indicator of chlorophyll content in plant leaves. This new chlorophyll fluorescence ratio indicates chlorophyll levels with high precision- the error in chlorophyll prediction over a wide range of chlorophyll content (from 41 to 675 mg m−2) was less than 40 mg m−2. The technique was tested and validated in three plant species: beech (Fagus sylvatica L.), elm (Ulmus minor Miller), and wild vine (Parthenocissus tricuspidata L.).

Non-mevalonate isoprenoid biosynthesis: enzymes, genes and inhibitors

The essential steps of the novel non-mevalonate pathway of isopentenyl diphosphate and isoprenoid biosynthesis in plants are described. The first five enzymes and genes of this 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway are known. The herbicide fosmidomycin specifically blocks the second enzyme, the DOXP reductoisomerase. The DOXP/MEP pathway is also present in several pathogenic bacteria and the malaria parasite. Hence, all herbicides and inhibitors blocking this novel isoprenoid pathway in plants are also potential drugs against malaria and diseases caused by pathogenic bacteria.

Cloning and heterologous expression of a cDNA encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase of Arabidopsis thaliana.

Various plant isoprenoids are synthesized via the non‐mevalonate pathway of isopentenyl diphosphate formation. In this pathway, 1‐deoxy‐D‐xylulose 5‐phosphate (DOXP), the first intermediate, is transformed to 2‐C‐methyl‐D‐erythritol 4‐phosphate (MEP) by an enzyme which was recently cloned from Escherichia coli. In order to find a plant homologue of this 1‐deoxy‐D‐xylulose 5‐phosphate reductoisomerase (DXR) we cloned a cDNA fragment from Arabidopsis thaliana which has high homology to the E. coli DXR. By expression of this fragment in E. coli we could demonstrate that it encodes a protein which transforms DOXP to MEP. The antibiotic fosmidomycin specifically inhibits this DXR enzyme activity.